Nerve stimulation increases the concentration of ATP in the synaptic cleft, which can act as a neurotransmitter or as a presynaptic neuromodulator. Using the luciferin-luciferase assay, we observed that the extracellular concentration of ATP increased by 11-26 nM over a basal concentration of 6 nM, in a frequency dependent manner (1-5 Hz), in the adult rat phrenic nerve-hemidiaphragm preparation. This ATP release depends on nerve activity since it was abolished by tetrodotoxin (1 [mu]M) and is strictly dependent on the presence of extracellular calcium. However, more than half of this nerve-evoked release of ATP is derived from activated muscle fibres since the selective post-synaptic nicotinic receptor antagonist, [alpha]-bungarotoxin (1 [mu]M), inhibited by over 60% the evoked release of ATP. The presently observed post-synaptic release of ATP together with the previously reported lack of post-synaptic effects of ATP and to the ability of ATP to act as a presynaptic modulator open the possibility that ATP may behave as a retrograde messenger at this neuromuscular junction.